We have previously shown that dopaminergic and non-dopaminergic neurones in the early postnatal rat substantia nigra (SN) are labelled by antibodies raised against NR3A and NR3B NMDA receptor subunits. NR3 subunits, unlike NR2 subunits, form diheteromeric NMDA receptors that are gated solely by glycine rather than by glutamate and glycine. Glycine-activated NR1/NR3 receptors are non-selective cation channels which are excitatory, by contrast with the well-established inhibitory role of glycine-gated Cl- channel receptors. In order to determine whether SN neurones express functional glycine-activated NR1/NR3 NMDA receptors we have made whole-cell patch-clamp recordings from SN neurones in midbrain slices prepared from rats aged 5-16 days old (decapitated under halothane anaesthesia). SN neurones were voltage-clamped to -60 mV using a patch pipette filled with either a high [Cl- ] solution to set ECl at 0 mV, or a low [Cl-] solution to set ECl at -55 mV. Glycine (1mM) was applied from a picospritzer (10 p.s.i. for 1s) in the presence of a cocktail of inhibitors in the perfusion solution and in the picospritzer pipette (strychnine, 600 nM; D-AP5, 50 μM; picrotoxin, 50 μM; tetrodotoxin, 100nM) to block glycine-gated Cl- channels, NR2-containing NMDA receptors, GABAA receptors and action potential-dependent neurotransmitter release respectively. When ECl was set to 0 mV, glycine evoked an inward current of -2095 ± 697 pA (n = 4). When ECl was set to -55 mV to minimise Cl- conductances, a glycine-evoked inward current was still observed at -60 mV although this was smaller in amplitude (-387 ± 59 pA; n = 7). Glycine-activated outward currents were seen at +40 mV (2285 ± 355 pA; n = 4). Repeated applications of glycine (every 200s for 1200s) caused a decrease in the amplitude of glycine-activated currents (IGlycine(1200s)/IGlycine(200s) ratio, 0.19). These data are consistent with a glycine-activated excitatory cation current, suggesting that functional NR1/NR3 NMDA receptors may be present in developing SN neurones.